Hepatitis C virus (HCV) is a member of the Flaviviridae family of enveloped, positive-strand RNA viruses and constitutes the sole member of the genus Hepacivirus. HCV is a significant pathogen, with nearly 3% of the world's population, roughly 170 million people, persistently infected. Despite significant efforts, no vaccine exists for HCV, and current anti-viral therapeutics are inadequate for the majority of patients. The sequence diversity of HCV is complex, with virus organized into 6 distinct genotypes and over 30 subtypes. Additionally, HCV exists as many closely related viral sequences, termed quasi-species, in the infected individual, making specific pharmaceutical targeting of HCV proteins problematic due to the rapid evolution of escape mutants. It is increasingly evident that a broad collection of specific, pan-genotypic antiviral drugs targeting multiple essential viral functions, in addition to the current non-specific viral therapies, will be required for effective global control of HCV.
Considerable progress has been made in characterization of HCV in the decade and a half since it's discovery as the causative agent of non-A non-B hepatitis. Much of this knowledge is based on autonomously replicating HCV RNA replicons, as no cell culture system or small animal models exist for the propagation of HCV. The genome of HCV consists of a ˜9.6 kb RNA molecule containing a single open reading frame flanked by highly structured 5′ and 3′ non-translated regions (NTR). Viral RNA is translated to generate a polyprotein precursor via an internal ribosome entry site within the 5′ NTR. The polyprotein undergoes a complex series of cleavage events to yield the ten mature HCV proteins. Once processed, the HCV proteins assemble into the membrane associated RNA replicase that, in combination with undefined cellular factors, constitutes the machinery required for HCV RNA synthesis. Characterization of the replicase has defined the protease/helicase NS3 protein, the NS4A cofactor, the NS4B integral membrane protein, the NS5A protein, and the NS5B RNA dependent RNA polymerase as essential components. Crystal structures have been determined for at least some of these proteins, including NS3/NS4A and NS5B. These structures represent only two thirds of the proteins required for RNA replication, leaving an incomplete picture of the replicase. Nonetheless, these structures have allowed the development of exciting new HCV specific pharmaceuticals. To better understand the replicase and fuel further drug development, obtaining structural information for the remaining components of this machinery is of paramount importance. This is perhaps most evident for the enigmatic NS5A protein.
NS5A is a large (56-58 kDa) hydrophilic phosphoprotein of unknown function. The protein is post-translationally associated with ER derived membranes via an N-terminal amphipathic α-helix buried in the outer leaflet of the membrane. Recent work has proposed a three-domain model for NS5A organization and demonstrated the N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting the membrane anchor or the zinc-binding site are lethal for RNA replication, suggesting a direct role for NS5A in this process. Additionally, the ability of adaptive mutations in NS5A to greatly stimulate HCV replication suggests an important role for this protein as a regulator of RNA replication. NS5A phosphorylation varies with replication efficiency, suggesting an interaction of NS5A with a cellular kinase(s) regulates RNA replication. NS5A appears to directly interact with all of the viral components of the replicase, and the interaction with the viral polymerase modulates the activity of this enzyme in vitro. These data collectively suggest NS5A serves as both an active replicase component and a regulator of replication. The modulation of replication by NS5A has been proposed to represent a switch between the replicative form of the viral RNA and a form of RNA amenable to virion biogenesis. This switch may involve the release of NS5A from the replicase or the conformational alteration of NS5A such that it is then free to interact with a variety of cellular components. NS5A interacts with proteins involved in membrane morphology and vesicular transport, suggesting it alters membranes for replication or virion assembly. NS5A interacts with many proteins in mitogenic and apoptotic signalling, resulting in the modulation of cellular growth and survival that may be important for the development and maintenance of HCV persistent infection. The alteration of these pathways by NS5A may represent a causative link between HCV infection and hepatocellular carcinoma (HCC). NS5A may be involved in another aspect of long-term viral persistence, the escape from the cellular anti-viral response via the inhibition of the activity of the double stranded RNA dependent protein kinase (PKR). The interactions of NS5A with the host cell are far more complex than alluded to herein and the relevance of many of these interactions to HCV biology remains to be determined, but it is increasingly clear that NS5A is a dynamic manipulator of the viral replicase and host cell, with potentially far reaching consequences. At present, however, a more complete understanding of NS5A in these processes is hampered by the lack of a proven function for NS5A.